Project Abstract To form a functional nervous system, each neuron must find and connect to the appropriate synaptic targets. Axons must often travel long distances in search of their targets, directed by a variety of secreted and cell surface axon guidance molecules. These ligands steer axons by interacting with receptors on their growth cones and altering cell behavior through changes in cytoskeletal architecture, cell adhesion, and transcription. The complex trajectories that axons take to reach their destinations are often broken up into smaller segments, which are punctuated by intermediate targets or ?choice points.? In bilateral organisms, the midline is a vital choice point for a subset of CNS neurons, called commissural neurons, whose axons must cross it to integrate information between the left and right halves of the body. Midline crossing is necessary for normal motor and cognitive function and defects in this vital process can result in a wide variety of neurodevelopmental disorders. Midline crossing is controlled by a combination of attractive and repulsive cues secreted by midline glia. Commissural neurons are initially sensitive to attractive cues such as Netrin, which draw them to the midline. After crossing, however, they become responsive to repulsive cues such as Slit, which facilitate exit from the midline and prevent re-crossing. Pre-crossing commissural neurons must prevent premature responsiveness to these repulsive cues and do this, in part, by downregulating the Slit receptor Roundabout (Robo). Drosophila downregulate Robo surface expression via the trafficking receptor Commissureless (Comm), which shunts newly-synthesized Robo directly from the Trans-Golgi Network to the endolysosomal system and decreases Robo protein levels. Two PY motifs in the cytoplasmic tail of Comm are necessary for its ability to perform these tasks. As PY motifs are known binding sites for Nedd4-family HECT ligases, this strongly suggests that Comm downregulates Robo by interacting with intracellular ubiquitination machinery. The precise mechanistic relationship between Comm and ubiquitin ligases, however, has not been explored in great detail. In this proposal, we seek to test the hypothesis that Comm acts as an adaptor protein that brings E3 ligases into close proximity to Robo, and that ubiquitination of Robo targets it to the endolysosomal system for degradation. In aim one, we seek to elucidate the requirement for ubiquitin to traffic the Comm-Robo complex to endo/lysosomes, facilitate Robo degradation, and promote midline crossing. In aim two, we will investigate the biochemical and genetic interactions between Comm and the three Drosophila Nedd4 family HECT ligases (Nedd4, Su(dx), and Smurf). In summary, the proposed work will provide deeper insight into the mechanisms regulating surface expression of axon guidance receptors during the development of neural circuits.